Craniofacial abnormalities, which are primarily the result of abnormal craniofacial skeletal formation, are one of the most abundant human disorders. For instance, cleft palate occurs at a rate of 1 out of 1,000 newborns. To identify the primary cause of craniofacial abnormalities and establish a foundation for future therapeutic applications, it is critical to identify the signaling pathways that are involved in regulation of craniofacial skeletal development, and to determine their functional roles. We recently discovered a novel family of cysteine-rich, secretory proteins, R-spondin, which consists of four members in both humans and mice. We and others found that the R-spondin proteins are specific ligands for the Frizzled8 and LRP6 receptors and activate canonical Wnt signaling, leading to induction of beta-catenin-dependent gene expression. Gene expression analysis and a preliminary phenotype characterization of R-spondin2 null mutant mice strongly suggest that R-spondin2 is a novel genetic factor for cleft palate, and that it plays a key role in the development of the first branchial arch and its derived skeletal structures. The goal of proposed research is to elucidate the regulatory roles and signaling mechanisms of the R-spondin2 in craniofacial development. We propose: (i) to determine the in vivo role of the R-spondin2 gene in craniofacial development by analyzing molecular and cellular defects in R-spondin2 gene-targeted mutant mice, (ii) to determine whether R-spondin2 signaling in craniofacial development is mediated via the canonical Wnt signaling pathway, and (iii) to determine the regulatory roles of R-spondin in the noncanonical Wnt signaling pathway in cell culture model. We anticipate that our research will advance our understanding of the role of R-spondin2 and Wnt signaling in craniofacial development. PUBLIC HEALTH RELEVANCE: Many human craniofacial abnormalities are caused by perturbation of the specific signaling pathways that regulate craniofacial skeletal development. Information produced from this study will provide the foundation for future design and development of R-spondin-based therapeutic tools applicable to prevention and cure of human craniofacial skeletal diseases.